Conventional flow cytometers cause a number of particles such as cells to flow in a straight line in aqueous suspension. These particles are analyzed using a hydrodynamic method, whereby light is radiated onto particles flowing through a flow channel, thereby detecting the scattered light and fluorescent light from the particles and converting it to electronic signals for analysis. An important feature of the flow cytometer makes it possible to quickly analyze many particles at one time.
In FIGS. 4 and 5, such a conventional flow cytometer is shown.
Flow cell 34 has a flow channel 34a which has a sheath liquid together with particles subject to analysis flowing therethrough. In the flow channel 34a, a number of particles move through in a straight line caused by flowing of a sheath liquid based on a hydrodynamic method. Mount 32 supports flow cell 34 and is mounted to a bench 31 through adjusting mechanism 33. The flow cell 34 is movable in the x and y directions by adjusting screws 33x and 33y of the adjusting mechanism 33 respectively. Light focusing lens 44 focuses a beam L0 from a laser (not shown) onto particles traveling through the flow channel 34a of the flow cell 34, thereby causing the particles to radiate two types of light, namely scattered light and fluorescent light.
A forward scattering light detecting assembly 35 is located in the forward direction of the beam L0 (x-axis direction). The light detecting assembly 35 includes mount 43, light path tube 36, lens 37, and a light detector container 39. Light path tube 36 is supported by mount 43. One end of light path tube 36 close to flow cell 34 has lens 37 while the other end is close to light detector container 39. In light detector container 39, pinhole 40 and light detector 41 are contained. Pinhole 40 together with light detector 41 are movable in the y and z axes by means of adjustment screws 39y and 39z respectively. Scattered light L1 produced from particles collected through lens 37, with its background light or noise eliminated through pinhole 40, is finally received by light detector 41 and converted into electronic signals. Beam blocker 42 prevents beam L0 from getting into light detector 41.
A right angle light detecting assembly 45 is disposed in the right angle direction (y-axis) with respect to the propagating direction of beam L0. The right angle light detecting assembly 45 includes light path tube 46, lenses 47a and 47b, light detector container 49 and so on. Lenses 47a and 47b are disposed at one end of light path tube 46, while its other end is inserted into detector container 49. In light detector container 49, pinhole 50 and light detector 51 are disposed. Light L2 produced from particles collected through lenses 47a and 47b, with background noises eliminated through pinhole 50, is then received by light detector 51 and converted into electronic signals.
In conventional flow cytometers, flow cell 34 and forward light scattering detector 35 must be separately adjusted which takes much time to complete, especially when light axis lo of light beam L0 is not in line with light axis m of the forward scattering light detecting assembly 35. FIG. 3 shows a detailed depiction of these problems. In general, light beam L0 is a bell curve with a center light axis lo. Accordingly, if a particle p is on the light axis lo, the most intensified forward scattered light is received. Referring to FIG. 3c, a particle p flowing through a center of flow channel 34a is on both the light axis lo and light axis m of the forward scattered light detecting assembly 35, thereby showing the most optimal condition with light axis lo in line with axis m. On the contrary, in FIG. 3a, light axis lo is not in line with light axis m. Particle p is located on light axis m but light axis lo is not in line with particle p. In this case, flow cell 34 must be moved in the y-axis plus direction using adjustment screw 33y. So, when flow cell 34 is moved in the y-axis plus direction in order to locate the particle p on the light axis lo, the particle p is deviated from light axis m which means the focusing point Q of the forward scattered light L1 is also deviated from light axis m, thereby reducing the amount of light entering light detector 41 and lowering output signals as shown in FIG. 3b. Accordingly, adjustment screw 39y is operated to move pinhole 40 and light detector 41 in the y-axis minus direction.
However, if flow cell 34 is moved erroneously in the y-axis minus direction as in FIG. 3a, the light intensity received by the particle p decreases, thereby leading to a reduction of the output signals of light detector 41. For this reason, it is very difficult to judge whether flow cell 34 correctly moved based on the output signal of light detector 41. Therefore, in the actual practical light axis adjustment of flow cell 34, the slight movement of pinhole 40 and light detector 41 must be repeated to raise the output signal of light detector 41 every time flow cell 34 is slightly moved, so that light axis lo and light axis m are kept in line with each other. In addition, the width of light beam L0 and flow channel 21a is generally less than 0.2 millimeters, thereby requiring skillful manipulation of pinhole 40 and light detector 41 for light axis adjustment.